Project Summary/Abstract Osteoarthritis will affect 50 million Americans by 2020. The disease process is characterized by the aberrant gene expression and the inability of cartilage chondrocytes to repair the extracellular matrix leading to cartilage degeneration. CD-RAP is a small cartilage-characteristic protein that is co-expressed in cartilage with type II collagen and aggrecan. It is a compact gene and co-expressed with type IIB collagen in chondrogenesis, making it an excellent model for study of chondrocyte-specific gene expression. Over the past four years, we have extended our knowledge of a single gene to the analysis of the transcriptional regulation of the chondrocyte phenotype. Computational and experimental methods were combined to analyze groups of co- expressed genes and define common regulatory domains of genes expressed in articular cartilage. The current proposal will focus on extending these analyses to define transcriptional regulatory networks that predominantly operate in and define articular cartilage development (as opposed to the growth plate which results in bone formation). As new transcription or signaling factors arise, specific mechanisms of activity will be deciphered using our CD-RAP and COL2A1 gene models or cell culture. The specific aims of the proposal are: (1) Determine the mechanism of transcriptional regulation of the CD-RAP gene by the E-box proteins [unreadable]EF-1/USF1/USF2, C/EBPss, and HMGA. (2) Define the role of transcription factor networks in differentiation of cartilage from stem cells (3) Define the role of transcriptional regulation and signaling in a model for early osteoarthritis and (4) Arising from our preliminary experiments in Specific Aim 3, investigate the role of the intracellular regulator of EGF and FGF signaling, Sprouty-4, in chondrogenesis and osteoarthritis. The experimental approach will focus on (1) using genome-wide expression analyses to determine co-regulated genes, (2) screening for expression of transcription factors using a 1700 oligonucleotide array, (3) computational analyses for transcription factor binding domains of the promoters of co-regulated genes, and (4) confirmation of mechanistic function on the specific cartilage genes, CD-RAP and COL2A1. These studies will apply powerful techniques combining experimental and computational approaches to look beyond candidate genes to elucidate regulatory mechanisms critical for developing strategies to control chondrogenesis during development, in tissue engineering and repair, and ultimately to help find biological methods to control osteoarthritis.